Accurate determination of DNA content in single cell nuclei stained with Hoechst 33258 fluorochrome at high salt concentration

Araki, Tsutomu; Yamamoto, Akira; Yamada, Masa-oki

doi:10.1007/bf00492587

Cited by 58 publications

(41 citation statements)

References 17 publications

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“…In general, the error introduced by assuming 2 to be 2/3 is ϳ10% when donor-acceptor pairs possess anisotropy values less than 0.3 (44). This has been shown to be the case for BODIPY in several bilayers of differing fluidity (41) and HOC bound to DNA (45).…”

Section: Discussionmentioning

confidence: 99%

The Structural Organization of Cationic Lipid-DNA Complexes

Wiethoff¹,

Gill²,

Koe³

et al. 2002

Journal of Biological Chemistry

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The interaction of cationic liposomes with supercoiled plasmid DNA results in a major rearrangement of each component to form compact multilamellar structures comprised of alternating layers of two-dimensional arrays of DNA sandwiched between lipid bilayers. Fluorescence resonance energy transfer was used to estimate the distance of closest approach of DNA to the lipid bilayers in these complexes. The effect of several compositional variables on this distance, including the ratio of cationic lipid to DNA, and the charge density, intrinsic curvature, and fluidity of the lipid bilayer were examined. Additionally, the effect of ionic strength was studied. For complexes prepared at or above a 3:1 charge ratio (؉/؊), the observed distance of closest approach was found to be in agreement with the intercalation of DNA between lipid bilayers. As the charge ratio was decreased, a monotonic increase in the distance was observed with a maximum observed at 0.5:1. Correlations between differences in the proximity of DNA to the lipid bilayer and the hydrodynamic size of the complexes were also found. A model based on these observations and previous reports suggests the formation of discrete populations of complexes below a charge ratio of 0.5:1 and above 3:1. The structure of the negatively charged complexes is consistent with DNA extending from the surface of the particles, whereas those possessing excess positive charge were multilamellar aggregates with the DNA effectively condensed between lipid bilayers. Complexes between these two states consist of weighted fractions of these two species.

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Section: Discussionmentioning

confidence: 99%

The Structural Organization of Cationic Lipid-DNA Complexes

Wiethoff¹,

Gill²,

Koe³

et al. 2002

Journal of Biological Chemistry

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“…At 12 h after the release, the medium was again supplemented with nocodazole to prevent cells from passing thorough mitosis into the following G1 phase. At 22 h after the release, the injected cells were identified by immunofluorescent staining against GST and their nuclear DNA content was quantitatively evaluated by comparing nuclear Hoechst 33258 staining with that in uninjected cells (Araki et al, 1987).The nuclear DNA content of cells that had been microinjected with GST or wild-type GST-HsCdc6 was similar to that of uninjected cells at all times tested ( Figure 6A, open symbols). The nuclear DNA content of cells that had been microinjected with GST-HsCdc6-5X was approximately one half that of uninjected cells at each time point tested, indicating that the mutant form of HsCdc6 lacking the N-terminal Cdk phosphorylation sites prevented the G1/S transition ( Figure 6A, filled circles).…”

mentioning

confidence: 88%

Section: Hscdc6 Phosphorylation Site Mutants Block Chromosomal Dna Rementioning

confidence: 99%

“…To quantify the nuclear DNA content, cells that had been stained with rabbit polyclonal anti-GST antibody and the FITC-conjugated goat anti-rabbit secondary antibody (see above) were incubated for 1 h in 10 mM Tris-HCl (pH 7.5) containing 1 M NaCl and 2 M Hoechst 33258 fluorochrome at room temperature (Araki et al, 1987). Images of fluorescent cells were captured at 63ϫ magnification using a digital camera (CCD camera, model C 4880; Hamamatsu Phototonics, Bridgewater, NJ).…”

Section: Quantification Of Nuclear Dnamentioning

confidence: 99%

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Mutation of Cyclin/cdk Phosphorylation Sites in HsCdc6 Disrupts a Late Step in Initiation of DNA Replication in Human Cells

Herbig

Griffith

Fanning

2000

MBoC

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Cyclin-dependent kinases (Cdk) are essential for promoting the initiation of DNA replication, presumably by phosphorylating key regulatory proteins that are involved in triggering the G1/S transition. Human Cdc6 (HsCdc6), a protein required for initiation of DNA replication, is phosphorylated by Cdk in vitro and in vivo. Here we report that HsCdc6 with mutations at potential Cdk phosphorylation sites was poorly phosphorylated in vitro by Cdk, but retained all other biochemical activities of the wild-type protein tested. Microinjection of mutant HsCdc6 proteins into human cells blocked initiation of DNA replication or slowed S phase progression. The inhibitory effect of mutant HsCdc6 was lost at the G1/S transition, indicating that phosphorylation of HsCdc6 by Cdk is critical for a late step in initiation of DNA replication in human cells. INTRODUCTIONIn eukaryotes, the cell division cycle is regulated by the periodic activation and inactivation of cyclin-dependent kinases (Cdk), which are thought to phosphorylate protein substrates that are involved in driving cell cycle transitions (reviewed in Sherr, 1996;Roberts, 1999). The transition from G1 to S phase of the mammalian cell cycle requires the activity of cyclin E/Cdk2 and cyclin A/Cdk2. Cyclin E is expressed in middle to late G1 (Dulic et al., 1992, Koff et al., 1992, whereas cyclin A is first expressed at the G1/S transition of the cell cycle (Girard et al., 1991;Pagano et al., 1992;Zindy et al., 1992). Microinjection of anticyclin E or anticyclin A antibodies into human cells or expression of antisense cyclin A RNA, inhibits initiation of DNA replication (Girard et al., 1991;Pagano et al., 1992;Zindy et al., 1992;Tsai et al., 1993;Ohtsubo et al., 1995). Furthermore, enhanced levels of cyclins A and E accelerate the G1/S transition in vivo (Resnitzky et al., 1994(Resnitzky et al., , 1995 and in vitro (Krude et al., 1997).The initiation of DNA replication in eukaryotes requires the stepwise assembly of a multiprotein complex, called the prereplicative complex (pre-RC), on replicator elements in the chromatin (reviewed in Dutta and Bell, 1997;Newlon, 1997). Studies in yeast and in Xenopus have determined that a six-subunit complex, called origin recognition complex (ORC), serves to nucleate this assembly. In G1 of the cell cycle, the Cdc6 protein promotes the loading of the minichromosome maintenance proteins (MCMs) onto chromatin (Coleman et al., 1996;Aparicio et al., 1997;Donovan et al., 1997;Tanaka et al., 1997). This reaction requires an intact Walker A or Walker B motif in Cdc6 (Perkins and Diffley, 1998;Wang et al., 1999;Weinreich et al., 1999) and presumably involves direct physical interaction between Cdc6 and ORC (Li and Herskowitz, 1993;Liang et al., 1995;Mizushima et al., 2000). Although the mechanism of pre-RC assembly in human cells has not yet been characterized, all of the components of the pre-RC identified so far in yeast appear to be conserved in humans.A growing body of evidence indicates that components of the pre-RC are Cdk substrates who...

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“…Cell apoptosis and cell nucleus morphology were detected using the method of hoechst 33258 staining (Araki et al, 1987;Yao et al, 2006). Briefly, the cells were stained by Hoechst 33258 (1 µg/mL) at room temperature in dark for 15 min.…”

Section: Morphological Assessment Of Cell Apoptosis By Hoechst 33258 mentioning

confidence: 99%

Anti-inflammatory and Anticancer Activities of Ethanol Extract of Pendulous Monkshood Root in vitro

Huang¹,

Ren²,

Li³

et al. 2013

Asian Pacific Journal of Cancer Prevention

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Accurate determination of DNA content in single cell nuclei stained with Hoechst 33258 fluorochrome at high salt concentration

Cited by 58 publications

References 17 publications

The Structural Organization of Cationic Lipid-DNA Complexes

The Structural Organization of Cationic Lipid-DNA Complexes

Mutation of Cyclin/cdk Phosphorylation Sites in HsCdc6 Disrupts a Late Step in Initiation of DNA Replication in Human Cells

Anti-inflammatory and Anticancer Activities of Ethanol Extract of Pendulous Monkshood Root in vitro

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